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PURPOSE: To evaluate the unknown genetic causes of teratozoospermia, and determine the pathogenicity of candidate variants. METHODS: A primary infertile patient and his family members were recruited in the West China Second University Hospital of Sichuan University. Whole-exome sequencing was performed to identify causative genes in a man with teratozoospermia. Immunofluorescence staining and western blotting were applied to assess the pathogenicity of the identified variant. Intracytoplasmic sperm injection (ICSI) was used to assist fertilization for the patient with teratozoospermia. RESULTS: We performed whole-exome sequencing (WES) and detected a novel homozygous frameshift mutation of c.335_336del [p.E112Vfs*3] in DNAJB13 on a primary infertile male patient. Intriguingly, we identified abnormal sperm morphology in this patient, with recurrent respiratory infections and chronic cough. Furthermore, we confirmed that this mutation resulted in negative effects on DNAJB13 expression in the spermatozoa of the affected individual, causing ultrastructural defects in his sperm. Remarkably, our staining revealed that DNAJB13 was expressed in the cytoplasm of primary germ cells and in the flagella of spermatids during spermiogenesis in humans and mice. Finally, we are the first group to report a favorable prognosis using ICSI for a patient carrying this DNAJB13 mutation. CONCLUSION: Our study revealed a novel homozygous frameshift mutation of c.335_336del [p.E112Vfs*3] in DNAJB13 involved in teratozoospermia phenotype. Our study greatly expands the spectrum of limited DNAJB13 mutations, and is expected to provide a better understanding of genetic counseling diagnoses and subsequent treatment of male infertility. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-022-02431-1.

Two DL models were developed using radiograph-level annotations (yes or no disease) and fine-grained lesion-level annotations (lesion bounding boxes), respectively named CheXNet and CheXDet. The models' internal classification performance and lesion localization performance were compared on a testing set (n=2,922), external classification performance was compared on NIH-Google (n=4,376) and PadChest (n=24,536) datasets, and external lesion localization performance was compared on NIH-ChestX-ray14 dataset (n=880). The models were also compared to radiologists on a subset of the internal testing set (n=496). Given sufficient training data, both models performed comparably to radiologists. CheXDet achieved significant improvement for external classification, such as in classifying fracture on NIH-Google (CheXDet area under the ROC curve [AUC]: 0.67, CheXNet AUC: 0.51; p, Radiology: Artificial Intelligence

This study aims at investigating drivers' risk perception ability. To achieve this objective, the risk sensitivity and risk judgment thresholds of drivers of different ages were calculated. Five scenarios of intersections with risks were established for the driving simulator experiment. The driving behavior data of fourteen younger drivers and fourteen elderly drivers during the risky event and the subjective evaluation of risk by an expert driver were collected. The expert driver's conflict degree and subjective feeling were combined to classify the risk level of driving scene; then, fuzzy signal detection was used to calculate the driver's risk sensitivity (d') and judgment threshold (β). The β with the greatest difference was selected for cluster analysis and drivers were divided into four types according to the threshold. Finally, a driver classification discriminant model was constructed based on Fisher discriminant analysis. The results show that d' and β of younger drivers are both better than those of elderly drivers, younger drivers can detect and respond risks in time, while elderly drivers need be closer to risks and have intuitive feelings to make judgments. The results of the cluster analysis showed that younger drivers account for a large proportion of the sensitive type, indicating that younger drivers can find risks more sensitively in risk scenarios than elderly drivers, while elderly drivers easily ignore risks due to physical and psychological weakness. The correlation analysis showed that age, saccade amplitude and heart rate are the main factors that affect the risk judgment threshold.

The proportion of older drivers has increased with the aging population. In order to improve the driving behavior and safety of older drivers, we aim to analyze behavior differences between older and younger drivers and then study an improvement strategy based on the older drivers’ behavioral characteristics. Older drivers’ behaviors can be enhanced through training, thereby improving driving safety. Simulated scenarios for behavior analysis and training are constructed for drivers who are recruited from the general driving population. Data on the drivers’ eye movement, physiological and psychological conditions, operation behavior, and vehicle status are collected and analyzed. The theory of planned behavior is adopted to construct a driving behavior enhancement training model for older drivers. Finally, a structural equation model is developed to comprehend the relationship between training level, driver characteristics, and traffic safety. The ability and speed of older drivers to obtain traffic information is worse than those of young and middle-aged drivers, and the vehicle control capability of older drivers has a larger volatility. The driving behavior training model can improve older drivers’ driving stability and safety, as follows: the positive effect of training on driving behavioral improvement is larger than the negative effect of aging; the negative effect of training level on dangerous driving tendency is larger than the positive effect of driver’s aging. The driving behavior of older drivers should be improved for the safety and stability of driving operations through the PNE (perceived-norm-execution) model. The relationship between training level, driving behavior characteristics, and traffic safety is discussed using the structural equation model, and results show that the training can improve the effect of the drivers’ age on the characteristics of driving behavior, and that older drivers tend to decrease dangerous driving tendencies.

Generalized nucleus segmentation techniques can contribute greatly to reducing the time to develop and validate visual biomarkers for new digital pathology datasets. We summarize the results of MoNuSeg 2018 Challenge whose objective was to develop generalizable nuclei segmentation techniques in digital pathology. The challenge was an official satellite event of the MICCAI 2018 conference in which 32 teams with more than 80 participants from geographically diverse institutes participated. Contestants were given a training set with 30 images from seven organs with annotations of 21,623 individual nuclei. A test dataset with 14 images taken from seven organs, including two organs that did not appear in the training set was released without annotations. Entries were evaluated based on average aggregated Jaccard index (AJI) on the test set to prioritize accurate instance segmentation as opposed to mere semantic segmentation. More than half the teams that completed the challenge outperformed a previous baseline. Among the trends observed that contributed to increased accuracy were the use of color normalization as well as heavy data augmentation. Additionally, fully convolutional networks inspired by variants of U-Net, FCN, and Mask-RCNN were popularly used, typically based on ResNet or VGG base architectures. Watershed segmentation on predicted semantic segmentation maps was a popular post-processing strategy. Several of the top techniques compared favorably to an individual human annotator and can be used with confidence for nuclear morphometrics.

Colorectal cancer (CRC) grading is typically carried out by assessing the degree of gland formation within histology images. To do this, it is important to consider the overall tissue micro-environment by assessing the cell-level information along with the morphology of the gland. However, current automated methods for CRC grading typically utilise small image patches and therefore fail to incorporate the entire tissue micro-architecture for grading purposes. To overcome the challenges of CRC grading, we present a novel cell-graph convolutional neural network (CGC-Net) that converts each large histology image into a graph, where each node is represented by a nucleus within the original image and cellular interactions are denoted as edges between these nodes according to node similarity. The CGC-Net utilises nuclear appearance features in addition to the spatial location of nodes to further boost the performance of the algorithm. To enable nodes to fuse multi-scale information, we introduce Adaptive GraphSage, which is a graph convolution technique that combines multi-level features in a data-driven way. Furthermore, to deal with redundancy in the graph, we propose a sampling technique that removes nodes in areas of dense nuclear activity. We show that modeling the image as a graph enables us to effectively consider a much larger image (around 16$\times$ larger) than traditional patch-based approaches and model the complex structure of the tissue micro-environment. We construct cell graphs with an average of over 3,000 nodes on a large CRC histology image dataset and report state-of-the-art results as compared to recent patch-based as well as contextual patch-based techniques, demonstrating the effectiveness of our method., Comment: Accepted in ICCVW 2019 (Visual Recognition for Medical Images)

A ring-shaped traveling wave ultrasonic motor with a suspension stator is proposed for improving output power density. Piezoelectric stacks working in d33 mode are involved in the stator, whereas piezoelectric plates employed in classic ring-shaped traveling wave motors often work in d31 mode. Spring-mass systems are used to suspend the stator from the base that supports the motor and provide preload force. The volume of piezoelectric material and the power density of the motor can be easily increased through the suspension structure. Large vibration amplitude of the stator can be generated on the coupling surface. Simulink analysis and finite element analysis are conducted to verify the theory and determine the optimum parameters of the suspension stator with four piezoelectric stacks. The maximum free vibration amplitude of 4.25 µm of the stator is observed, which is nearly 4.7 times of that without suspension. The prototype can work at a maximum no-load speed of 62 rpm and produce a stall torque of 49.5 mN m under a driving signal of 30 Vpp when the mass block is 0.30 g. Furthermore, the higher output power can be expected because more extra stacks can be added in this suspension structure.

RpoS, the stationary-phase sigma factor of Escherichia coli , is responsible for increased transcription of an array of genes when cells enter stationary phase and under certain stress conditions. RpoS is rapidly degraded during exponential phase and much more slowly during stationary phase; the resulting changes in RpoS accumulation play an important role in providing differential expression of RpoS-dependent gene expression. It has previously been shown that rapid degradation of RpoS during exponential growth depends on RssB (also called SprE and MviA), a protein with homology to the family of response regulators, and on the ClpXP protease. We find that RssB regulation of proteolysis does not extend to another ClpXP substrate, bacteriophage lambda O protein, suggesting that RssB acts on the specific substrate RpoS rather than on the protease. In addition, the activity of RpoS is down-regulated by RssB when degradation is blocked. In cells blocked for RpoS degradation by a mutation in clpP , cells devoid of RssB show a four- to fivefold-higher activity of an RpoS-dependent reporter fusion than cells overproducing RssB. Therefore, RssB allows specific environmental regulation of RpoS accumulation and may also modulate activity. The regulation of degradation provides an irreversible switch, while the regulation of activity may provide a second, presumably reversible level of control.

Regulated degradation of RpoS requires RssB and ClpXP protease. Mutations in hns increase both RpoS synthesis and stability, causing a twofold increase in synthesis and almost complete stabilization of RpoS, independent of effects on synthesis and independent of phosphorylation of RssB. This suggests that H-NS regulates an RssB inhibitor or inhibitors.

Proteases play a pivotal role in metabolic regulation. They degrade many key regulatory proteins as well as abnormal proteins and thereby let cells adjust to varied environmental conditions. In Escherichia coli, five ATP-dependent proteases have been characterized: Lon (or La), ClpAP, ClpXP, ClpYQ (also called HslUV), and FtsH (5, 6). Lon was the first of these proteases to be identified and purified. E. coli mutants defective in the lon protease display a pleiotropic phenotype (7). Two of these phenotypes are sensitivity to DNA-damaging agents and overexpression of capsular polysaccharide. These two phenotypes are due to stabilization of two substrates of the Lon protease, SulA and RcsA, respectively. SulA, an inhibitor of cell division, is induced upon DNA damage (10). In lon+ cells, SulA is degraded very rapidly, with a half-life of less than 2 min; in lon mutant cells, stabilization of SulA results in irreversible filamentation and subsequent cell death (11, 26). RcsA, an unstable positive regulator of genes involved in colanic acid capsular polysaccharide, is degraded by the Lon protease and has a half-life of 5 min in lon+ cells. In lon mutant cells, the accumulation of RcsA (with a half-life of more than 20 min) leads to overexpression of capsular polysaccharide (33, 34). Although lon mutants have profound effects on RcsA and SulA turnover, the residual degradation of SulA (22) and appreciable turnover of RcsA (34) in lon mutant cells led to the prediction that other proteases that can degrade these Lon substrates must exist. The work described here was originally begun to identify this protease(s) and led to the isolation of strains with a phenotype referred to as Alp (alternate Lon protease) (35). Alp strains had the UV resistance and low capsule synthesis of a lon+ strain but were devoid of Lon. Alp activity was originally detected in a screen of a plasmid genomic library for plasmids that would suppress lon mutant phenotypes. Trempy and Gottesman (35) found that an activity (then designated Alp and now called AlpA), when present in a multicopy plasmid, can suppress both lon mutant phenotypes of sensitivity to DNA-damaging agents and overexpression of capsular polysaccharide. Rapid proteolysis of SulA, as was found in lon+ cells, was found in Δlon cells that carried the AlpA plasmid. This activity, like Lon and Clp, is energy dependent. Further studies revealed that the AlpA open reading frame affected protease activity indirectly, by leading to the inactivation of ssrA (17, 36). Inactivation of ssrA by an insertion mutation mimicked the effect of the Alp plasmid. Thus, the product of the ssrA gene inhibited the expression of the Alp protease activity. At the time the original Alp work was done, ssrA was known to encode a small stable RNA (10Sa RNA) of unknown function. It has now been shown to encode tmRNA, a molecule with both tRNA-like and mRNA-like properties that acts cotranslationally to add an 11-amino-acid tag to the C termini of proteins whose translation is interrupted or stalled in the absence of a nonsense codon (14). The resulting tagged proteins are then sensitive to degradation by the ClpAP and ClpXP proteases (8). Thus, at first glance, one would expect ssrA mutants, unable to add a degradation signal to polypeptides on stalled ribosomes, to decrease the degradation of certain tagged proteins, contrary to our initial identification of the mutants as promoting a protease activity able to substitute for Lon. Even more puzzling than the role of ssrA was the observation that for full and stable expression of Alp activity, cells also had to harbor a multicopy plasmid carrying a kanamycin resistance element (designated AHA for Alp helper activity) (17). Since the original publications on the relationship of Alp to ssrA, we and others have shown that ClpYQ, an ATP-dependent protease induced under heat shock conditions, is capable of degrading SulA and RcsA (13, 16, 20, 42). Full suppression of the capsule overproduction or of the UV sensitivity of a lon mutant required overproduction of ClpYQ (42). However, the suppression of capsule synthesis in lon strains, a hallmark of the activity we originally identified genetically and called Alp, was not reversed by mutations in clpY or clpQ (42) and therefore remained unidentified. The experiments described here were begun with the aim of understanding the basis for the kanamycin resistance gene role and the mechanism of action of Alp. Mutations that bypassed the kanamycin resistance requirement were sought. We report here that a novel allele of dnaJ has the capacity to substitute for AHA and, in fact, can suppress lon phenotypes even in ssrA+ hosts. Studies of this mutant and new information on tmRNA led us to a new understanding of the Alp phenomenon as two separate suppression events, the ClpYQ-dependent degradation of SulA and the inactivation of the capsule synthesis pathway under heat shock stress.

All organisms use rapid degradation of specific proteins as one way to tightly control important regulatory factors and developmental switches. Regulation of biological functions by proteolysis requires accurate substrate recognition at a precise time. In eukaryotes, fidelity of protein degradation depends on ubiquitin-tagging, a process that occurs in multicomponent complexes involving regulatory factors and various ubiquitin pathway enzymes. The ubiquitinated proteins are then targeted to the proteasome for degradation (for review, see Voges et al. 1999). In Escherichia coli, an example of substrate tagging as a means for regulating proteolysis is the degradation of incomplete proteins made from truncated mRNAs. In this case, short peptides coded by the SsrA RNA are added cotranslationally to polypeptides that have become stalled on ribosomes, and the tagged proteins are then degraded (Keiler et al. 1996; Gottesman et al. 1998; Roche and Sauer 1999). In general, E. coli proteins regulated by degradation interact directly with the proteases themselves, with each of the five known E. coli ATP-dependent proteases degrading a different but somewhat overlapping set of substrates (Gottesman 1996; Wickner et al. 1999). For many of the highly unstable E. coli proteins, degradation is rapid under all conditions, and synthesis is tightly controlled. However, the activity of some proteins in E. coli is controlled by regulated degradation. For example, σ32 (RpoH), the heat shock sigma factor, is rapidly degraded under normal growth conditions by the AAA protease, FtsH, in a reaction modulated by the DnaJ/DnaK/GrpE chaperone system. During heat shock, σ32 is transiently stabilized, and this stabilization results in the rapid increase in the synthesis of the heat shock proteins (Yura and Nakahigashi 1999). The stationary phase sigma factor, σS (RpoS), is another example of a protein whose activity is controlled by regulated proteolysis. σS promotes expression of more than 50 genes involved in responses to many stresses, including starvation, osmotic stress, acid shock, cold shock, heat shock, and oxidative damage, as well as the transition to stationary phase (Loewen and Hengge-Aronis 1994; Hengge-Aronis 2000). Although σS is present at very low levels during exponential cell growth, owing largely to its rapid degradation (half-life of ∼2 min), its stability increases ∼10-fold following transition to stationary phase or other stress treatments. Regulated degradation plays a major role in determining the amount of σS in the cell, but σS accumulation is also regulated at the transcriptional and translational levels (Lange and Hengge-Aronis 1994). The protease responsible for σS turnover in exponentially growing cells is ClpXP (Schweder et al. 1996), an ATP-dependent protease consisting of a regulatory component, ClpX, and a proteolytic component, ClpP (Gottesman et al. 1993; Wojtkowiak et al. 1993). Degradation of σS requires an additional protein, RssB (Regulator of Sigma S; also referred to as SprE in E. coli, MviA in Salmonella, and ExpM in Erwinia) that is homologous to response regulator proteins (Bearson et al. 1996; Muffler et al. 1996b; Pratt and Silhavy 1996; Andersson et al. 1999). Genetic evidence shows that RssB is required for σS degradation but not for another ClpXP substrate, λ O, which indicates that RssB specifically targets σS for degradation (Zhou and Gottesman 1998). One of the hallmarks of the response regulator component of two-component signal transduction systems in prokaryotes is the presence of a conserved aspartate that is phosphorylated by the cognate sensor component. The N-terminal domain of RssB contains this conserved aspartate, although a cognate sensor protein has not been identified. RssB, like many other response regulators, is phosphorylated by acetyl phosphate in vitro (Bouche et al. 1998). In addition, phosphorylated RssB forms a stable complex with σS in vitro (Becker et al. 1999). In this report we investigate RssB-regulated degradation of σS by reconstituting the pathway of σS degradation in vitro with purified RssB and ClpXP. We discovered that RssB acts directly and catalytically in stimulating degradation of σS by ClpXP in a reaction requiring acetyl phosphate and ATP. We have isolated and characterized subassemblies of the degradation machinery including σS–RssB, σS–RssB–ClpX, and σS–RssB–ClpXP complexes, and suggest a probable pathway for the degradation of σS.

The ClpYQ (HslUV) ATP-dependent protease of Escherichia coli consists of an ATPase subunit closely related to the Clp ATPases and a protease component related to those found in the eukaryotic proteasome. We found that this protease has a substrate specificity overlapping that of the Lon protease, another ATP-dependent protease in which a single subunit contains both the proteolytic active site and the ATPase. Lon is responsible for the degradation of the cell division inhibitor SulA; lon mutants are UV sensitive, due to the stabilization of SulA. lon mutants are also mucoid, due to the stabilization of another Lon substrate, the positive regulator of capsule transcription, RcsA. The overproduction of ClpYQ suppresses both of these phenotypes, and the suppression of UV sensitivity is accompanied by a restoration of the rapid degradation of SulA. Inactivation of the chromosomal copy of clpY or clpQ leads to further stabilization of SulA in a lon mutant but not in lon + cells. While either lon , lon clpY , or lon clpQ mutants are UV sensitive at low temperatures, at elevated temperatures the lon mutant loses its UV sensitivity, while the double mutants do not. Therefore, the degradation of SulA by ClpYQ at elevated temperatures is sufficient to lead to UV resistance. Thus, a protease with a structure and an active site different from those of Lon is capable of recognizing and degrading two different Lon substrates and appears to act as a backup for Lon under certain conditions.

Certain proteins and protein fragments in Escherichia coli are modified by carboxy-terminal addition of an 11-residue peptide tag (Tu et al. 1995). This tagging process requires functional SsrA RNA (10Sa RNA), which encodes the last 10 residues of the peptide (Tu et al. 1995) and results in rapid degradation of the tagged protein by carboxy-terminal-specific proteases (Keiler et al. 1996). SsrA-mediated tagging of proteins translated from defective messenger RNAs lacking termination codons has been demonstrated, and a model in which SsrA functions both as a tRNA and an mRNA has been proposed (Keiler et al. 1996). The 363-nucleotide SsrA RNA has sequences that form a tRNA-like structure and has been shown to be chargeable with alanine (Komine et al. 1994; Williams and Bartel 1996; Felden et al. 1997). In the model proposed by Keiler et al., when ribosomes stall at the 3′ end of the damaged message, SsrA charged with alanine binds to the ribosome like a tRNA and contributes the alanine to the idle nascent chain. Translation then switches from the mRNA to a small open reading frame (ORF) in SsrA that encodes the carboxy-terminal degradation peptide. This system provides both a method to avoid the accumulation of ribosomes stalled at the end of defective messages and a general quality-control mechanism that allows the cell to rid itself of incomplete protein fragments that might have inappropriate cellular activities. Cells devoid of SsrA RNA grow more slowly and show a certain degree of temperature sensitivity (Oh and Apirion 1991; Komine et al. 1994; Trempy et al. 1994). The involvement of carboxy-terminal amino-acid sequences in targeting proteins for rapid degradation was recognized before the discovery of the SsrA-tagging system (Bowie and Sauer 1989; Parsell et al. 1990), and a periplasmic protease (Tsp or Prc) that degrades protein substrates in a carboxy-terminal-specific manner was purified and characterized (Silber et al. 1992). The carboxy-terminal substrate sequences recognized by Tsp are similar to those of the SsrA tag (Keiler et al. 1995; Tu et al. 1995), and Tsp is responsible for degradation of SsrA-tagged proteins that are exported to the periplasm (Keiler et al. 1996). Cytoplasmic proteins with carboxy-terminal degradation sequences, however, are still proteolyzed rapidly in cells lacking Tsp (Silber and Sauer 1994; Keiler et al. 1996), indicating that other proteases must be responsible for carboxy-terminal-specific degradation of proteins in the bacterial cytoplasm. Essentially all cytoplasmic degradation in prokaryotes, archaea, and eukaryotes is energy-dependent. E. coli, for example, has at least five ATP-dependent proteases [Lon (La); HflB (FtsH); ClpAP; ClpXP; and ClpYQ (HslUV)] (for review, see Gottesman 1996). These enzymes appear to have distinct substrate preferences, as a mutation in a single protease gene is often sufficient to stabilize a specific unstable protein. For example, mutations in Lon lead to stabilization of the N protein of bacteriophage λ, the SulA and RcsA proteins of E. coli, and the CcdA protein of the episomal F factor. HflB appears to be responsible for degradation of the cII protein of λ and the heat-shock σ factor RpoH (Herman et al. 1993, 1995). The principal substrates for ClpYQ degradation have not yet been identified, although this two-component protease has been implicated in degradation of both Lon subtsrates and HflB substrates in vivo (Missiakas et al. 1996; Kanemori et al. 1997; Khattar 1997; W.-F. Wu and S. Gottesman, unpubl.). ClpAP and ClpXP are two-component proteases that share a common proteolytic subunit, ClpP, but have different ATPase regulatory subunits, ClpA or ClpX. Proteins stabilized by mutations in clpX but not in clpA include λ O, phage Mu repressor variants, and the stationary-phase σ factor, RpoS; clpA but not clpX mutants stabilize certain LacZ fusion proteins and the MazE protein. ClpB, an ATPase with extensive sequence similarity to ClpA, has not thus far been demonstrated to have a direct role in proteolysis but may act as a chaperone (Squires and Squires 1992). In the studies presented here, we show that intracellular degradation of variants of the amino-terminal domain of λ repressor containing the SsrA peptide tag is dramatically reduced in cells lacking ClpP or lacking both ClpX and ClpA, and is somewhat reduced in cells lacking ClpX or ClpA only. Purified ClpXP and purified ClpAP degrade SsrA-tagged protein substrates in vitro, suggesting that these ATP-dependent enzymes are directly responsible for degradation of SsrA-tagged proteins in the bacterial cytoplasm.